Approximately 60% of currently marketed drugs are orally administered formulations whose clinical efficacy critically depends on the absorption from the small intestine (SMI). However, currently available in vitro intestinal models rely predominantly on cancer cell lines that do not recapitulate the 3D microenvironment of the small intestine. Likewise, animal models often fail short in predicting in vivo human outcomes of candidate drugs. During Phase I, we successfully developed a promising small intestine drug permeation model based on an in vitro, organotypic tissue comprised of human SMI cells. Characterization of the in vitro SMI tissues showed good correspondence to native human tissue in terms of histology, transepithelial electrical resistance, and structural features. The utility of the SMI model for drug permeation studies was demonstrated. In vitro SMI permeability data strongly correlated with in vivo human absorption data (r2 = 0.87 - 0.95) whereas data from the widely-used Caco-2 cell line model was less predictive (r2 = 0.81). Permeability data also demonstrated that efflux transporters were functional in the SMI in vitro tissue and inhibition studies showed that the SMI tissue will likely be useful to study drug-drug interactions. An economic analysis of the in vitro model showed significant advantages versus comparable rodent bioavailability studies. The ultimate goal of this project is produce a validated, biologically relevant organotypic SMI model that predicts intestinal drug absorption/bioavailability of orally administered drugs. The human in vivo-like characteristics of the SMI model and its capacity to measure drug absorption, metabolism, and drug-drug interactions make it a superior tool to existing in vitro and ex-vivo methods. In the proposed application, we will finalize a drug permeation and metabolism prediction model. Reproducibility of the model will be determined and transferability of the in vitro assay methods to other laboratories will be demonstrated. Successful completion of these Phase 2 goals will result in an extremely useful model for early preclinical drug screening. The human primary cell based SMI tissue model will improve pharmacokinetic analysis of new drug formulations, accelerate drug development, and reduce the ever- increasing development cost of drugs.